The mucosa is a primary site for pathogen transmission, but injected vaccines induce poor mucosal immunity. Mucosal dendritic cell subsets in the buccal tissue can induce both immunity and tolerance to antigens. The proposed research focuses on inducing mucosal immunity by activating mucosal dendritic cells using recombinant outer membrane vesicles (rOMVs). Over the past decade, our research team has designed and developed rOMVs to induce robust Th1/Th2 immune responses to peptide antigens. In more recent and yet unpublished work, our team has discovered a way to activate and drive the maturation of DCs. Perhaps most exciting is that we quantified the penetration depth of rOMVs in the buccal mucosal tissue of the pig and show that the ~100 nm vesicles penetrate the tissue to depths consistent with the population of nave myeloid dendritic cells (>100 m). This rOMV-based technology, therefore, has a profile consistent with a mucosally-deliverable vaccine, specifically via buccal tissue. The objective of the research is to illustrate and establish the uniqueness of the rOMV approach as a method for buccal vaccine delivery. By combining the use of subunit epitopes with the tissue penetration of rOMVs, we will evaluate the hypothesis that the administration of these rOMVs to the buccal mucosa will induce the activation of nave DCs and induce a strong and balanced Th1/Th2 immune response. The hypothesis will be evaluated through the completion of the following Aims. Aim 1: to quantify the immune response to a ?weak? antigen to determine the efficacy and potency of the rOMVs. Our previous work used green fluorescent protein (GFP) as an effective model of a ?weak? antigen that demonstrated the potency of rOMVs administered subcutaneously. We will use the same approach to quantify both the humoral and cellular responses to GFP delivered by rOMVs to the buccal mucosa of pigs (the closest animal model to the human buccal mucosa). The results will be compared to the same formulation administered subcutaneously (i.e., benchmark) in order to correlate the efficacy of the buccal route of administration to a traditional route of administration. Aim 2: to quantify the immune response to the hemagglutinin (HA) antigen of influenza to determine the protective immunity conferred by the rOMVs. In previous work we used a sequence of HA delivered by subcutaneous injection of rOMVs in mice to quantify the level of protective immunity, as measured by the hemagglutinin inhibition assay and the antibody neutralization analysis. We will use the same approach to quantify both the humoral and cellular responses to HA delivered by rOMVs to the buccal mucosa of pigs. The results will be compared to the same formulation administered subcutaneously (i.e., benchmark) in order to correlate the efficacy of the buccal route of administration to a traditional route of administration. The Cornell- based research team of Putnam (rOMV vaccine design) and Chang (swine immunity) combines the labs of two experts who collectively have the skill to create an effective, buccally administered vaccine delivery platform that is potentially applicable to a wide range of mucosally-transmitted pathogens.